Bacterial oxygenases: potential, features and new investigation methods.

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UNIVERSITY OF INSUBRIA

Ph.D. School in Biological and Medical Sciences

Ph.D. Program in

Analysis Protection and Management of Biodiversity

- XXIV Course -

BACTERIAL OXYGENASES:

FEATURES, POTENTIAL

AND NEW INVESTIGATION METHODS

Supervisor: Prof. Paola Barbieri

Coordinator: Prof. Marco Saroglia

Ph.D. thesis of

Luca Bianchi

2008-2011

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Abstract

Bacterial oxygenases, the enzymes involved in the breakdown pathways of aromatic and aliphatic hydrocarbons, have aroused great interest within the scientific community for their potential applications in a number of different fields: environmental (bioremediation and biomonitoring), biotechnological (green chemistry) and biomedical (drug production).

The aims of this study were 1) to clone new bacterial oxygenases using conventional methods and further develop new screening techniques and 2) to assess the ability of the newly-cloned enzyme(s), of toluene/o-xilene monooxygenase (ToMO) and of some of its mutants, to exploit various types of aromatic substrate on which the activity of these enzymes has never previously been assessed.

Using conventional techniques, Pseudomonas sp. N1 genes coding for naphthalene dioxygenase (NDO) and Sphingobium sp. PhS genes, putatively coding for Phenanthrene dioxygenase (PhDO), were cloned; of the two enzymes only NDO appeared to be functional. ToMO is an enzyme complex formed from six different subunits, ABCDEF;

subunit A (TouA) is part of the terminal hydroxylase and contains the active site. In this study, in addition to wild type enzymes, the TouA mutants D211A and D211A/E214G, previously created by this laboratory, and TouA E214G, already described in the literature, were also used.

The ability of ToMO and TouA mutants, and of NDO to hydroxylate different aromatic compounds other than their natural substrates (toluene and naphthalene respectively) was evaluated by biotransformation assays using, as substrates, the following molecules: 1,2,3- trimethoxybenzene, anisole, benzophenone, bibenzyl, biphenyl, nitrobenzene, quinoline, and trans-stilbene. These compounds were chosen for the environmental, biotechnological and pharmacological importance of their hydroxylated derivatives. To this end, ToMO, TouA mutants and NDO were expressed in the heterologous host E. coli JM109. For both enzymes, the products of bioconversion were identified by GC-MS and, using the appropriate standard of reference, quantified by HPLC analysis.

ToMO proved active in all substrates tested. The mutant E214G, like the wild type, was capable of oxidising all the compounds tested: in almost all the biotransformations both the mutant form and the wild type behaved in the same way in that they both produced the same isomers in similar proportions. In terms of catalytic efficiency, the mutant E214G was found to be most powerful: the mutants D211A and D211A/E214G were capable of

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hydroxylating only a few of the substrates used (1,2,3-trimethoxybenzene, anisole, benzophenone, biphenyl) with very low yields. However, considering the fact that strains expressing the mutants D211A e D211A/E214G showed impaired growth and expression problems, probably attributable to unwanted mutations introduced during mutagenesis, these data cannot be considered definitive.

The ability of NDO to perform dihydroxylation processes was confirmed but, in addition, monohydroxylation reactions (quinoline) and dealkylation reactions (anisole and 1,2,3- trimethoxybenzene) were observed. The quantitative analysis of the biotransformation products showed that the enzyme efficiency decreased with the increasing steric footprint of the substrate. The failure of nitrobenzene to be transformed suggests that, in addition to steric footprint, other factors such as the presence of deactivating substituent groups plays a key role in the catalytic process of this enzyme.

Both NDO and ToMO have shown a good potential in the field of biotransformation and further studies could lead to the production of more useful and efficient enzyme variants.

During an internship at the University of Granada in Spain, a new protocol, based on the SIGEX (Substrate Induced Gene Expression) system, was developed to screen bacterial genomic libraries. This method, which is normally used to screen metagenomic libraries, was adapted to analyse libraries created from a single bacterial genome. With this technique it is possible to perform a rapid screening on the basis of a signal emitted from a reporter gene included in the construct used in creating the library. Using a previously constructed library, this approach allowed, in a short time, 60 clones to be isolated that had a high probability of containing naphthalene-dependent regulatory genes. The sequencing of the obtained clones, a necessary step in order to validate the protocol, in currently in progress.

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1. Introduction

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1. Introduction

1.1. The metabolic potential of microorganisms

The disposal of aromatic and aliphatic organic compounds, whether naturally occurring or man made, is a problem of global concern with important political and economical implications.

The chemical industry, with the production of polymers, combustibles, solvents, pesticides and pharmaceuticals, has released and continues to release into the environment vast amounts of 'xenobiotics' (from the Greek xenos; stranger), that is to say, synthetic compounds with a different structure to that of a naturally occurring molecule. In the broadest sense, the term can also be used for those compounds that, although of a natural origin, are not normally present in any appreciable quantities in the biosphere such as, for example, petroleum. Ecological disasters, where the accidental loss of compounds resistant to biodegradation (e.g. the oil spill from the tanker Haven in the Bay of Genova in 1991;

the sinking of the tanker Prestige off the coast of Galicia in 2004), of course, do not help the above situation. In this scenario of environmental emergencies, associated with development that has now become unsustainable, the world of microorganisms plays a crucial role [1].

Microorganisms, with their vast natural repertoire of metabolic and genetic diversity, the result of a long and complex evolutionary process, constitute a huge reservoir of biodiversity that has been, and still has, a major role in the maintaining balance within the biosphere [2]. Over the course of evolution, bacteria have developed a great ability to adapt to different environmental conditions, managing to exploit many different chemical compounds as a source of carbon and energy [3]. Nevertheless, while molecules that have always been present in the environment, such as monocyclic aromatic hydrocarbons, are degraded by numerous microorganisms, xenobiotic compounds, as well as many polycyclic aromatic hydrocarbons (PAHs), are often resistant to biodegradation: the former because they only came into contact with microorganisms around one hundred years ago, too short a time period to allow the evolution of metabolic pathways that are capable of degrading them, the latter because of their structural complexity.

In relatively recent times, particular bacterial strains have been isolated that are capable of using complex aliphatic and aromatic hydrocarbons as a source of carbon and energy, converting them into intermediates of their central metabolism [4, 5]. The microorganisms already described, of which the numbers are continually increasing, have aroused great interest in the scientific community for their potential application in different areas of

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biotechnology. These include bio-remediation of contaminated sites, biotransformation of toxic compounds into less harmful products or products of high industrial value (green chemistry), and the development of bio-sensors for the monitoring of pollutants. The range of substrates on which these microorganisms are able to grow is extensive and includes both toxic compounds and those that are difficult to degrade, such as benzene, toluene, ethylbenzene, xylenes (BTEX), a number of substituted phenols, polycyclic aromatic hydrocarbons (PAHs), and chlorinated organic compounds [4, 6, 7].

In recent years, the presence of a large number of contaminated sites has led to the rapid development of soil remediation technologies. Among them, the one that has aroused most interest is biological degradation. The choice of technique to be used for the remediation of a contaminated site is determined by many factors according to the nature of the pollutants, the site in question, the technology itself, and the economic and legal evaluations. In the case of contamination by hydrocarbons or organic substances, ex-situ biological treatments represent an effective and low-cost intervention. Where microbial communities capable of carrying out the breakdown process are already present within the site, it is possible to enrich, in-situ, the species responsible for this process, mainly through the addition of limiting inorganic nutrients and oxygen (biostimulation) [3]. In areas lacking naturally- occurring microorganisms capable of metabolising xenobiotic compounds, the introduction of isolated biodegrading strains from other sites (bioaugmentation) is necessary.

Bioaugmentation is conditional on the ability of the selected strains to survive in indigenous microbial communities; often the microorganisms used in this process are derived from selective enrichment cultures and, therefore, are not representative of the indigenous microflora of the recipient site. For this reason, after being inoculated on site, they survive for only a brief period of time as they are unable to compete effectively for nutrients [8].

Bacterial strains specialised in the metabolism of such compounds can be produced in the laboratory, both naturally by selecting, in the presence of contaminants, mutants or recombinant strains, and by using genetic engineering techniques that allow the desired characteristics to be transferred into the appropriate host and/or the implementation of targeted modifications. The use of recombinant bacterial strains is, however, limited to confined areas (bioreactors) because current European legislation does not allow the introduction of genetically modified microorganisms into the natural environment [9].

Bioremediation procedures, compared to traditional chemical decontamination methods, are environmentally friendly, exhibit a low energy consumption and are low in cost [10]

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1. Introduction

Furthermore, the study of these molecules' catabolic processes has shown that microorganisms and enzymes involved in the breakdown stage, are also tools that can be used effectively in a different biotechnological area, namely bioconversion. The partial degradation of a large number of organic compounds often leads to the formation of intermediates with high added value[11]. In addition, the enzymes involved in one or more stages of the bacterial degradation pathways usually also show activity on compounds that are structurally similar to natural substrates and are, therefore, available for the formation of derivative compounds that are of great industrial and biotechnological interest [12, 13].

Therefore the ability of bacteria to adapt themselves to be able to exploit new chemical compounds, such as growth substrates, is a subject of great and growing interest, not only for the potential applications in the field of bioremediation but also in bioconversion [14].

Industrial processes for the synthesis of fine chemicals, that involve purified microorganisms or enzymes, are steadily increasing [15]. It is estimated that, by the year 2050, 30% of the production processes of the entire global chemical industry will be based on biocatalysis [16]. The environmental impact of biocatalytic processes is extremely small, as they require milder reaction conditions compared to traditional chemical processes, they reduce the need for, potentially dangerous, strong acids and bases, they generally require low concentrations of metals and, usually they don't require the use of solvents. They also involve the formation of fewer undesirable products, reducing the energetic demand and increasing the safety of the process [11].

Finally, it should be remembered that microorganisms and enzymes involved in the breakdown stages of metabolism can also lead to the development of biosensors for the detection of common environmental pollutants. One example is represented by the genetically modified strain P. fluorescens HK44 [17, 18]. This strain is able to detect the presence of an environmental contaminant by emitting a bioluminescent signal, allowing it to be used as an effective in-situ monitoring tool [18, 19].

It is evident that the use of specific enzymes in biotechnological applications, both for bioremediation and for industrial biocatalysis requires, as a prerequisite, an extensive knowledge of the genetic and biochemical characteristics of the microbial catabolic pathways involved.

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1.2. Microbial metabolic pathways

The breakdown of aromatic compounds can occur both aerobically and anaerobically, through energetic metabolic processes, or through co-metabolic processes in which the microorganisms receive neither energy nor a source of carbon. This latter process is particularly important in the breakdown of mixtures of aromatic and PAH compounds of a high molecular weight [20]. Although these compounds have long been considered recalcitrant to breakdown under anoxic conditions, it has been observed that, in fact, breakdown does happen under these conditions, albeit in a slow and inefficient way.

Certain members of the genus Clostridium are, in fact, able to promote the dehalogenation of polychlorinated biphenyls in anaerobic conditions [21]; another example is the bacterial strain EbS7 isolated from marine sediment samples and belonging to the subclass δ of the proteobacteria, which can, anaerobically, completely mineralise ethylbenzene by linking its oxidisation to the reduction of sulphates [22].

In aerobic conditions, however, the breakdown of aromatic compounds is much quicker and more efficient due to the ability to activate stable molecules using molecular oxygen.

The aerobic catabolic pathways can be divided into the following functional segments [23]:

the processing of substituent groups; transformation of aromatic substrates into dehydroxylated intermediates; the opening of the ring and production of linear compounds containing 2 or 4 carbon atoms; entry of the products into the tricarboxylic acid (TCA) cycle .

Although microorganisms possess a wide variety of enzymes for the initial attack on a number of different compounds, the catabolic pathways usually converge: the substrates are converted into a small number of key intermediates that are subsequently metabolised by a single central pathway. This results in simpler control units and a more efficient metabolic process.

The initial stage of oxidation of an organic compound is catalysed by an oxygenase that, in the presence of molecular oxygen, is able to attack the aromatic ring of the molecule, leading to the formation of dihydroxylated compounds such as catechols, gentisate and protocatechuate (upper pathway). These dihydroxylated intermediates can be cleaved by ring-opening oxygenases to give linear compounds (lower pathway) that can then enter the TCA cycle. The aromatic ring can be opened by the meta cleavage pathway, characterised by the extradiol opening of the ring, or by the ortho cleavage pathway, in which the ring is opened intradiol, that is between the two hydroxyl groups (Fig. 1.1) [25].

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1. Introduction

In the last few decades, more than 100 species have been isolated, belonging to different genera, that are able to breakdown these aromatic compounds [4]. The largest number and the most specialised species belong to the genus Pseudomonas [26, 27]; to a lesser extent the genera Ralstonia, Mycobacterium, Polaromonas, Streptomyces, Sphingomonas, amongst others, also possess similar breakdown capabilities [28, 29].

Fig. 1.1. General outline of the breakdown of aromatic compounds.

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Bacteria belonging to the genus Pseudomonas represent the most remarkable group of microorganisms capable of using, in aerobic conditions, many aromatic and aliphatic compounds as the sole source of carbon and energy. These are the ubiquitous gram- negative bacteria, present in soil and water and capable of establishing symbiotic relationships with higher organisms. These bacteria are capable of breaking down a wide variety of highly aromatic pollutants including BTEX, PAHs, and substituted phenols, as well as extremely cytotoxic compounds such as polychlorinated biphenyls (PCBs). This great metabolic versatility, attributable to the low substrate specificity of the catabolic enzymes, allows these microorganisms to break down structurally related molecules using the same pathways. Frequently, the genes coding for enzymes involved in the catabolism of aromatic compounds are found on mobile genetic elements, such as transposons and plasmids, which facilitate horizontal genetic transfer and the rapid adaptation of the microorganisms to new pollutants. Gene transfer, sometimes interspecific, mediated by processes such as conjugation and transduction, increases the genetic variability of microorganisms and favours the evolution of new catabolic pathways [30]. Recombination events lead to the creation of mosaics of catabolic operons, which can provide the microorganisms with completely new metabolic properties suitable for metabolising new molecules [31, 32, 33].

1.3. Bacterial oxygenases

Oxygenase enzymes play a key role in the breakdown of aliphatic and aromatic hydrocarbons. These enzymes are able to catalyse the introduction of oxygen into organic compounds from simple oxygen molecules (O₂) [23]. The types of oxygenase are of great importance to the industrial sector as they can be used in the chemical synthesis, “green chemistry”, of useful products in pharmaceutical and agrochemical fields [34]. The oxygenases can be divided, on the basis of the reaction mechanism, into monooxygenase and dioxygenase.

Monooxygenase catalyses the introduction of a hydroxyl group into the substrate, which can be an aromatic or aliphatic compound. In this reaction, the reduction of two oxygen atoms is seen: one reduced to a hydroxyl group, the other to a water molecule, all in conjunction with the oxidation of NAD(P)H. Monooxygenase is primarily involved in the

“upper pathway”.

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1. Introduction

In aerobic environments, dioxygenase catalyses both the initial NAD(P)H-dependent hydroxylation reaction of the aromatic ring (upper pathway), facilitating the simultaneous introduction of the two hydroxyl groups into the ring, and the reactions that lead to the opening of the aromatic ring (lower pathway) without the intervention of external reducing agents [35] (Fig. 1.2).

Enzymes belonging to the family of bacterial oxygenases are, often structurally similar, multi-component systems that show, however, notable differences in their substrate range and in the regio-specificity of oxygenation of the aromatic ring [36]. These systems are composed of multiple functional components organised into a short electron transfer chain from NAD(P)H to oxygen. Usually component I is an NAD(P)H-dependent flavoprotein oxyreductase, component II is a ferredoxin containing an iron sulphur centre, while component III is a terminal hydroxylase that contains the active site (usually a monoferric or diferric centre) (Fig. 1.3). The transport of electrons starts with the transfer of one (for monooxygenase) or two (for dioxygenase) electrons from NAD(P)H to the flavin, followed by the transfer of single electrons to the Fe-S centres then to iron on the terminal hydroxylase component, which is usually composed of large oligomeric proteins [35]. In

Fig. 1.2. a) Dioxygenase activation reactions of an aromatic ring incorporating two hydroxyl groups at the expense of O2 and NADH. b) and c) reactions involving dioxygenase opening the aromatic ring.

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some monooxygenase enzymes, additional subunits have been identified that are, apparently, indispensable for catalysis although their role is not always entirely clear.

1.4. Metabolism of naphthalene

Naphthalene is a bicyclic aromatic hydrocarbon that is commonly found in the environment and is often used as a model molecule for studying the breakdown pathways of PAHs as it is the simplest of its class. Many bacteria belonging to the genera Alcaligenes, Burkholderia, Mycobacterium, Polaromonas, Pseudomonas, Ralstonia, Rhodococcus, Sphingomonas, and Streptomyces have been isolated that use naphthalene as the sole source of carbon and energy [29]. One of the best characterised enzyme systems responsible for the breakdown of naphthalene is encoded by the plasmid NAH7 in Pseudomonas putida G7: NAH7 has two operons that contain the genes responsible for the breakdown of naphthalene [27].

In the first stage of naphthalene breakdown (Fig. 1.4), two groups of OH are introduced in positions 1 and 2 of the aromatic ring to form cis-(1R,2S)-dihydroxy-1,2- dihydroxynapthalene (Fig. 1.5) (cis-dihydrodiol of naphthalene) by a naphthalene dioxygenase (NDO). The subsequent stages proceed as shown in Figure 1.5, until catechol or gentisic acid is produced, which is further oxidised to obtain linear compounds that are able to enter the TCA cycle.

Fig. 1.3. Schematic representation of the components of a bacterial oxygenase.

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1. Introduction

In several cases, it has been observed that enzymes involved in the oxydation of naphthalene are able to break down phenanthrene and anthracene to 1-hydroxy-2- naphthoate and 2-hydroxy-3-naphthoate, respectively (Fig. 1.6). The catabolic pathways of other PAHs are essentially similar although, due to the high number of aromatic rings present, the number of steps required to obtain the linear compounds is increased.

Fig. 1.4. Initial oxidation of naphthalene to cis-1,2-dihydroxy-1,2- dihydroxyaphthalene by naphthalene dioxygenase.

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Fig. 1.5. General schematic of the breakdown pathway of naphthalene.

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1. Introduction

Fig. 1.6. Peripheral pathway for the catabolism of phenanthrene.

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1.5. NDO in strains of Pseudomonas

The sequencing and characterisation of genes coding for enzymes involved in the catabolism of naphthalene was made possible by isolating the plasmid NAH7 (83 kb) from P. putida G7 [37]. The catabolic genes are organised in two operons: one codes for upper pathway enzymes that are involved in the conversion of naphthalene into salicylate, while the second codes for lower pathway enzymes that are involved in the conversion of salicylate into intermediates of the TCA cycle. Finally, a third gene (nahR), located between the two operons but transcribed independently, encodes the transcriptional activator (NahR), that acts for both [38]. NahR is necessary for the high level of nah gene expression and for their induction by salicylate [39].

Plasmids bearing the genes for the catabolism of naphthalene (called plasmid NAH), as well as derivatives of pWW60 from P. putida NCIB9816, pDTG1 from P. putida NCIB 9816-4 and pKA1 from P. fluorescens 5R, have been found to be very similar to the NAH7 plasmid of the G7 strain. The nucleotide sequences of genes coding for upper pathway enzymes of naphthalene, isolated from various strains of Pseudomonas, are available in the databases: ndo of P. putida NCIB 9816, nah of P. putida G7 and NCIB 9816-4, dox of Pseudomonas sp. C18, pah of P. putida OUS82 and P. aeruginosa PaK1 and nah of P.

stutzeri AN10 [20]. The complete sequencing of these genes has been carried out for the strains OUS82, PaK1 and AN10; in other cases only partial sequencing was performed.

The genetic organisation and sequencing of upper pathway catabolic genes of these strains is, however, similar to that of nah genes in the plasmid NAH7 of the G7 strain (Fig. 1.7).

In strains of Pseudomonas, the first four ORFs, nahAaAbAcAd, code for the four subunits NhaAa/Ab/Ac/Ad composed of NDO: NhaAa and NhaAb, an oxyreductase flavoprotein and a ferrodoxin, provide a short electron transport system, formed by the oxidation of NAD(P)H. The two subunits NahAc and NahAd form the terminal hydroxylase complex (Fig. 1.8 a) in a α3 β3 configuration (Fig. 1.8 b). The catalytic site, comprising a monoferric centre, is located inside the α subunit. The nahB, nahF, nahC, nahE and nahD genes, that code for the enzymes involved in the later breakdown stages of naphthalene, are found below the nahAaAbAcAd cluster [40] (Fig. 1.9).

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1. Introduction

Genes coding for the enzymes involved in the PAH breakdown pathways of the Pseudomonadaceae bacterial family are usually organised in clusters and, in a few species, have already been isolated and cloned. The analysis of these sequences has allowed the identification of varying portions and highly conserved portions, the latter usually being located on the regions coding for the enzyme's active site. These conserved portions are useful because they can be used to design probes that can look for new oxygenases.

Research on new naphthalene dioxygenase is particularly interesting because it has been observed that enzymes with small differences in amino acid sequences have different substrate specificities.

Fig.1.7. Catabolic genes of the upper pathway of naphthalene in strains of Pseudomonas.

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.

Fig. 1.8. a) Schematic representation of NDO enzymatic complex . b) crystallographic structure of NDO hydroxylase

b a

Fig. 1.9. Genetic organisation of the nah chromosome region of P. stutzeri AN10.

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1. Introduction

1.6. Sphingomonadaceae

The breakdown of aromatic molecules often occurs due to an interaction between different microbial strains through the formation of real microbial consortia in which, by establishing synergistic relationships, each different member is involved in only one, or a few, stages of the catabolic process [41]. A number of strategies can promote the breakdown of recalcitrant compounds such as the formation of a biofilm, a highly organised biological system in which the bacterial cells, in sessile form, are organised into a functional community with a specific spatial architecture and strong adherence to the substrate [42]. A further strategy is the production and release of biosurfactants, surfactants that are able to increase the bioavailability of highly lipophilic substrates [43].

Members of the Sphingomonadaceae family, that have recently been isolated and show a certain level of genetic richness, are capable of metabolising even very heterogeneous mixtures of mono- and polyaromatic compounds. Some authors argue that Sphingomonadaceae has such a broad metabolic versatility because the genes involved in the early phases of PAH breakdown are not combined in a single catabolic operon, but are scattered throughout the genome, a feature that allows them to easily reorganise their enzyme system. This alternative organisation of genes involved in the breakdown pathways of aromatic compounds distinguishes them from other bacterial genera, such as Pseudomonadaceae, in which the genes coding for breakdown enzymes tend to be grouped into operon units [44]. Based on phylogenetic analysis, that includes the 16S rDNA test and chemotaxonomic analysis, the Sphingomonadaceae were divided into four genera:

Sphingomonas, Sphingobium, Novosphingobium, and Sphingopyxis [45]. A fifth genus, Sphingosinicella, was added only later [46]. Bacteria belonging to this family are part of the subgroup α-Proteobacteria, are Gram negative, aerobic and chemotrophic, and their principle characteristic is the presence a particular class of molecule, the glycosphingolipids (GSL) in the external membrane. GSLs occupy the same position as lipopolysaccharides (LPS) in other Gram negative bacteria and perform much the same function, as well as exhibiting a similar structure to that of biosurfactants. The carbohydrate chain of GSL is, however, shorter than that of LPS, making the cell membrane of these bacteria more hydrophobic [45]; many Sphingomonadaceae are also able to secrete anionic heteropolysaccharides, known as sphingans.

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1.6.1. Genes and enzymes for the catabolism of PAHs in Sphingomonadaceae

Members of the Sphingomonadaceae family are able to use a wide variety of aromatic compounds, including PAHs, as the sole source of carbon and energy. For example, Novosphingobium aromaticivorans F199, is able to break down toluene, xylene, p-cresol, biphenyl, naphthalene, dibenzothiophene, fluorene, salicylate e benzoate [47], while Sphingobium yanoikuyae B1 is able to grow on 1,2,4-trimethylbenzene, toluene, p- ethyltoluene, m- e p- xylene, biphenyl, naphthalene, phenanthrene and anthracene [48].

The ability of these microorganisms to breakdown polycyclic aromatic hydrocarbons is currently being studied in order to explain how these bacteria can use such a wide range of compounds.

The catabolic genes identified in the genus Pseudomonas can be located at the chromosome or plasmid level but, regardless of its position on the genome, they are grouped in clusters [44]. In contrast, the catabolic genes in Sphingomonadaceae are not grouped into operon units, but are scattered throughout the genome [49]. Some authors support the hypothesis that these catabolic sequences encountered a certain amount of evolutionary restriction that reduced the genetic exchange between Sphingomonadaceae and other bacterial genera [44].

The sequencing of the catabolic plasmid pNL1 (184 kb) from N. aromaticivorans F199 [50] has identified at least 79 genes grouped into 15 clusters, that code for the enzymes associated with the breakdown and transport of aromatic compounds. In respect of the oxygenase involved in the first step of the catabolism of phenanthrene, there appears to be a common gene arrangement in which the catalytic components (bphA1a-f and bphA2a-f) appear to interact with a single set of electron transporters (bphA3 and bphA4). Many parts of the DNA sequence in the pNL1 regions coding for genes that are responsible for the catabolism of aromatic are similar to those of S. yanoikuyae B1, S. paucimobilis Q1, Sphingomonas sp. HV3, Sphingomonas sp. DJ77, S. paucimobilis TNE12 e Sphingobium sp. P2 [51]. These findings have suggested that an unusual arrangement of different genes, belonging to different catabolic pathways, is characteristic of the Sphingomonadaceae and that such organisation could contribute to their high versatility. In fact, if the amino acid similarity of the enzymes involved in the different catabolic pathways of aromatic compounds and the different organisation of the corresponding gene sequences are

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1. Introduction

considered, it's possible to hypothesise that the various gene clusters can modify their position and orientation within the genome, combining themselves into modules that can be added to other oxygenase genes [32]. Although many functional analyses on gene expression for the breakdown of aromatics have been reported, the function of each original dioxygenase remains unclear [52].

1.7. Bacterial metabolism of toluene

The breakdown of toluene can occur through dioxygen reactions (via TOD), through the monooxygenation of the aromatic ring in various locations [53, 54, 55], or through the progressive oxidation of the methyl group (via TOL) [56].

In Pseudomonas putida F1, the breakdown of toluene starts with the oxidation of the benzene ring by a toluene dioxygenase (TDO) forming the intermediate cis-dihydrodiol, followed by dehydrogenation that produces d 3-methylcatechol (Fig. 1.10), which then opens in the meta position [57, 58]. The genes that code for this enzyme form part of the tod operon that contains the genes for toluene dioxygenase (tod C1C2BA). In Burkholderia cepacia G4 and Ralstonia pickettii PKO1, toluene 2-monooxygenase and toluene 3- monooxygenase catalyse the formation of 3- methylcatechol through two, subsequent, monooxygenation reactions; the first of which produces the phenolic, o-cresol and m-cresol intermediates, respectively [53, 55] (Fig. 1.10). In Pseudomonas mendocina KR1, the upper pathway for the breakdown of toluene, that combines the hydroxylation of the aromatic ring and the oxidation of the methyl group, involves toluene-4-monooxygenase (T4MO) [59]. T4MO converts the toluene to p-cresol through a monooxygenation reaction.

p-cresol is then oxidised, through subsequent transformations of the methyl group, to protocatechuic acid (the central intermediate of aromatic compound breakdown, together with catechol) that is finally mineralised via the orto-pathway (Fig. 1.11). In this particular case, given that it doesn't form catechol as the central intermediate, the aromatic ring opening involves another enzyme from the intradiolic dioxygenase family, the protocatechuate 3,4-dioxygenase. The dehydroxylated compounds produced by the reactions in the upper pathway are then broken down into intermediates that enter the Krebs cycle through the lower pathway.

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Fig. 1.10. Upper pathways for the breakdown of toluene in three different species Pseudomonas.

Fig. 1.11. Metabolism of toluene in Pseudomonas mendocina KR1.

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1. Introduction

1.7.1. Toluene/o-xilene monooxygenase in Pseudomonas sp. OX1

Pseudomonas sp. OX1 is one of the few microorganisms capable of breaking down toluene and o-xylene: this ability is due to the presence of an enzyme called toluene / o-xylene monooxygenase (ToMO). ToMO hydroxylates toluene and o-xylene, producing the corresponding phenols and is also responsible for the further hydroxylation of the phenolic intermediates in (di)methylcatechol [60]. Another characteristic of this bacteria, that contributes to its ability to adapt to polluted environments, is its resistance to organic and inorganic compounds containing mercury. The resistance to mercury is encoded by a plasmid, while the genes involved in the catabolism of hydrocarbons are located on the chromosome [61].

In the genome of Pseudomonas sp. OX1, the genes that code for toluene and o-xilene catabolic enzymes are organised into two operons: the first, called tou operon, codes for the ToMO complex; the second, called phe operon, contains the genes that code for Phenol Hydroxylase (PH), which is involved in the metabolism of phenol, and the genes coding for the enzymes of the lower pathway. The entire upper pathway includes, not only ToMO activity, but also that of PH. The breakdown process starts with the conversion of hydrocarbon into phenol, catalysed by ToMO. This event leads to the production of a mixture of o- m- and p-cresol from toluene and of 2,3- and 3,4-dimethylphenol from o- xylene; these intermediates are subsequently converted into 3,4-dimethylcatechol and 4,5- dimethylcatechol during a second monooxygenation reaction, catalysed by ToMO or PH (Fig.1.12). The breakdown starts, therefore, with two successive monooxygenation reactions, the first of which, catalysed by ToMO is not regiospecific, while the second, catalysed by ToMO or by PH, always occurs on a carbon in the ortho position in respect of the hydroxylated carbon [60]. The catechols produced by the monooxygenation reactions in the upper pathway are then broken down into intermediates that enter the Krebs cycle through the lower pathway.

The locus encoding ToMO maps in a 6 kb region of the Pseudomonas sp. OX1 chromosome, known as the tou (toluene o-xylene utilisation) locus: sequence analysis revealed the presence of a cluster of six ORFs called touABCDEF, each one coding for one of the subunits TouABCDEF which, together, constitute the whole enzyme complex (Fig.

1.13 a) [62].

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ToMO is composed of a flavoprotein oxyreductase (Tou F), a ferrodoxin Rieske-type (Tou C), a terminal hydroxylase complex (ToMO-H) whose formation combines the subunits Tou A, B and E in configuration (αβγ)2 (Fig. 1.14), and a catalytic effector (Tou D) (Fig.

1.13 b) [63]. The electrons, indispensable for the catalysis, derived from the oxidation of NAD(P)H and, through a short, two-component transport system (Tou F and Tou C), converge towards the Tou A subunit in which there is a Fe-O-Fe diferric centre, the active site of the enzyme. The Tou D subunit is thought to carry out a regulatory function by interaction directly with the catalytic subunit Tou A, thereby influencing the speed of the enzyme's reaction [63].

Fig. 1.12. Breakdown of toluene and o-xylene in Pseudomonas sp. OX1.

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1. Introduction

Fig. 1.13. a) Genetic organisation of the gene cluster encoding ToMO. b) Schematic representation of ToMO subunit organisation

Fig. 1.14. Ribbon representation of ToMO-H (crystallographic data).

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1.8. The contribution of molecular techniques and bioinformatics to the improvement of oxygenase systems

With modern genetic engineering, in a relatively short time you can create more stable and efficient enzymes that can be used by microorganisms for the purposes of degrading otherwise difficult compounds or for the production of intermediates of biotechnological interest. Several methods, including the construction of chimerical enzymes [64, 65] and random mutagenesis [66, 67] have been used by molecular biologists for the improvement of oxygenase systems.

These technologies, already in themselves advanced, are today of great support to bioinformatics; some resources, such as the NCBI, ExPASy and PDB portals, are available on the web and allow operations to be performed directly on-line starting from a simple consultation with a research database and comparison of nucleotide or protein sequences.

These indispensable tools are accompanied by much open source software specifically designed for the study of various types of molecule; software dedicated to 3D structural analysis, such as PyMol and SPDBV [68], and those dedicated to the study of intermolecular interactions, for example, Autodock 4.0 [69] are particularly useful for the study of enzymes. This latter software is capable of automatically performing docking simulations between a small compound and a candidate macromolecule, DNA, RNA or protein, where a crystallographic structure exists. On the basis of data obtained from in silico structural analysis conducted on ToMO-H [70], from automated docking simulations and from information obtained from the current literature regarding the TouA E214G mutant [71], two new TouA mutants, D211A and D211A/E214G, were created through site-specific mutagenesis by the laboratory of Environmental and Molecular Microbiology in the Department of Structural and Functional Biology at the University of Insubria - Varese [72]. These mutants were used in biotransformation tests on toluene and nitrobenzene for a preliminary functional characterisation. Both mutants proved to be less efficient than the wild type enzyme in the biotransformation of toluene.

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1. Introduction

1.9. Biotransformation in industrial processes

Biotransformation is a reaction accelerated by biological catalysts (biocatalysts), in most cases isolated enzymes or whole cells [73, 74]. In the production of fine chemicals, for some time the advantage of using purified enzymes as heterogeneous catalysts has been well understood. In a few cases, however, the use of purified proteins for bio-industrial applications suffers from problems of costs connected with their purification, as well as from the fact that, for oligomeric molecules, the purification of functional molecules is not always possible. This is particularly true for oxygenase enzymes where the use of whole cells is more economical both in terms of time and cost [11]. Enzymes are protected inside the cell and, therefore, exhibit a longer half-life. In addition, the reactions catalysed by oxygenase require, usually, a certain number of cofactors that become difficult to provide in vitro but that, on the contrary, the whole cells provide naturally. In general it is simpler and less costly to generate these cofactors in metabolically active cells [75].

Both isolated enzymes and whole cells are currently used in industrial synthesis processes and represent an area of ongoing research [76].

The catalytic activity exploited to achieve biotransformation is normally released by the normal physiological activity of the enzyme and is also used on substrates that are structurally very diverse from those found in nature and in conventional cell metabolism.

Many enzymes have a broad substrate specificity and modern protein engineering techniques allow a further widening of the range of biotransformable molecules [77].

Bioconversion techniques can offer a number of advantages that make them interesting as a complement to conventional synthesis techniques [78], in particular: high reaction selectivity (chemo-, regio-, stereoselectivity); mild and environmentally compatible reaction conditions; and reduced costs. Disadvantages, which often represent the main limitations of using biotransformations are: low stability of many biocatalysts in operational conditions; low yields; low numbers of commercially available biocatalysts;

and a high development time for the process on an industrial scale. A few of the above stated advantages, particularly those concerning the different forms of selectivity, are only potential advantages, and are not always guaranteed. Enzyme catalysis can be extremely stereoselective but, when using substrates that differ from natural ones, selectivity may be only partial. On the other hand, some of the disadvantages normally associated with biotransformation seem, nowadays, to be largely outdated. The selectivity, stability and activity of many biocatalysts in particular reaction conditions can now be improved

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through a variety of techniques such as: the selection of new enzymes through innovative screening techniques; improvement of the molecular biocatalysts using protein or metabolic engineering techniques; enzyme immobilisation; and finding the best reaction conditions (reaction engineering).

The production of molecules as single enantiomers or diastereoisomers is of growing importance, non only in the field of pharmaceuticals, but also in sectors of fine chemistry, food and materials [79]. The agencies that regulate the commercialisation of drugs now require separate toxicological studies for every impurity present in concentrations greater than 1%, including potential enantiomers or diastereoisomers of the active ingredient. It is therefore necessary to have available simple stereoselective methods (such as biotransformations) that allow enantiopure molecules to be obtained in limited quantities.

1.10. The potential of oxygenases in bioconversions

The hydroxylation reaction of natural aromatic compounds, catalysed by enzymes with oxygenase activity, has been identified as “potentially the most useful of all the biotransformations” [15]. Chemical processes that involve the introduction of an oxygen atom are often non-specific and at times require expensive and toxic reagents [80];

conversely, biocatalytic processes involving the use of oxygenase, in general, require milder reaction conditions.

The unique catalytic properties of oxygenases, such as the ability to hydroxylate unactivated carbon atoms in a regiospecific and/or enantiospecific way, gives these enzymes an indisputable value in the field of biosynthesis [75, 81]. Their ability to hydroxylate a number of hydrocarbons in specific positions and to generate specific enantiomers, also makes them useful for the biotransformation of compounds of pharmaceutical interest [11, 14]. In fact, aromatic di- and tri-hydroxylated compounds find applications in various fields such as the pharmaceutical industry, cosmetics and food; this is demonstrated, for example, by the global production of catechol, resorcinol, and hydroquinone, that reach levels estimated to be around 110,000 tonnes per year [82].

An example of a monooxygenase proposed as a biocatalyst for the synthesis of chiral epoxides is the styrene monooxygenase of P. fluorescens. These chiral compounds represent the starting molecules for the synthesis of a large number of fine chemicals [83].

Recently, site directed mutagenesis experiments were performed on the ToMO of P. sp.

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1. Introduction

OX1 to make it usable in the synthesis of high value compounds such as 4- methylresorcinol, methylhydroquinone, and pyrogallol [84]. One example, however, of a bacterial strain used as a breakdown biocatalyst is Pseudomonas putida ATCC 33015, a microorganism that is able to use p-xylene as the sole source of carbon and energy [85].

The ability of this bacterium has been studied in detail on the compound 2,5- dimethylpyrazine, where the oxidation product is an intermediate of important pharmaceutical synthesis processes; it is currently used in large-scale fermenters[11].

Even dioxygenase is used in biocatalysis. Toluene dioxygenase (TDO) is one such example: this enzyme is able to oxidise β-bromoethylbenzene leading to the formation of the corresponding cis-dihydrodiol, which is an important intermediate in the synthesis of codeine [86]. TDO is also used in the synthesis of Crixivan (Indinavir), an inhibitor of HIV protease. The key intermediate in the synthesis of this molecule is (-)-cis-(1S,2R)-1- aminoindan-2-ol; this compound can be synthesised directly from the enantiopure cis- (1S,2R)-dihydroxyindan, produced by TDO from indene. Another important biotransformation product, that has a broad use as a dye in the textile industry, is indigo, which is synthesised from indole; this substrate, recognised even by NDO, is oxidised to indoxyl, which demerises spontaneously to form indigo.

1.11. Metagenomic techniques for isolation of bacterial catabolic enzymes: the SIGEX method

Currently, research on genes coding for enzymes involved in the catabolism of PAH is based almost exclusively on the construction of genome libraries, followed by long and laborious screening and sequencing procedures. In recent years, as well as the genome of the individual microorganism, the attention of many research groups has focused on the

“genome” of entire microbial communities (metagenomics) present in the environment being studied such as, for example, soil and water contaminated with hydrocarbons.

Therefore, new techniques have been developed that can quickly identify, within a metagenomic library, catabolic genes that encode regulatory and structural elements.

One of the most advanced screening techniques of metagenomic libraries is based on the SIGEX (Substrate Induced Gene Expression) method [87]. This method exploits the fact that in bacteria many of the catabolic pathways present an operon-type genetic

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organisation, which sees regulatory genes topologically close to structural genes. Using, for the construction of the library, an “operon trap” cloning vector, which contains, under the polylinker, a gene reporter that encodes a fluorophore, it is possible to check whether a particular substrate is capable of acting as an inducer, i.e. promoting the transcription of the insert; if there is evidence of transcriptional activity, there would be a good chance that the clone in question contains a substrate-dependent regulatory element. For the selection and collection of cells following induction, flow-cytometry techniques are used – this is another peculiarity of this method as it is typically used for the analysis of eukaryotic cells.

The SIGEX protocol is based on four fundamental steps (Fig. 1.15): 1) construction of the metagenomic library using an “operon trap” vector containing a gene reporter (GFP); 2) treatment of the genomic library with the substrate of interest, for example a hydrocarbon belonging to the class PAH; 3) selection of cells that show fluorescence using flow- cytometry analysis; 4) isolation of GFP positive clones and sequencing of the fragments cloned in the relative constructs. The use of this technique allows, in a short time, clones containing substrate-dependent regulatory genes to be isolated. These elements can act as regulators of structural genes coding for enzymes that are involved in metabolism of the substrate inducer, or for enzymes responsible for the biosynthesis of useful breakdown molecules, for example, biosurfactants (bioemulsifiers). This powerful screening technique is perfectly suited to the research of genes encoding new bacterial oxygenase, as many of them have an operon-type genetic configuration (e.g. ToMO).

Fig. 1.15. Schematic representation of the SIGEX method.

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2. Aims

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Bacterial oxygenases are responsible for the activation of aromatic and aliphatic hydrocarbons by hydroxylation, which is the first step in their degradative pathways. These enzymes, due to their involvement in a variety of biocatalytic processes, are of great environmental and biotechnological interest.

The aims of this study include:

1) cloning new bacterial oxygenases using conventional culture methods (enrichment cultures prepared with PHAs) and developing new cloning techniques based on genomic library screening;

2) using biotransformation assays to carry out functional characterisations of the newly cloned enzymes, ToMO and the TouA D211A and D211A/E214G mutants, using, as substrates, compounds for which the activity of these enzymes has never previously been described and whose hydroxylated derivatives (1,2,3- trimethoxybenzene, anisole, benzophenone, bibenzyl, biphenyl, quinoline and trans-stilbene) are of great environmental, industrial and pharmacological interest.

In addition, with a view to achieving these objectives, it was resolved to standardise a biotransformation protocol, that produced good reaction yields for each substrate. Indeed, this condition was necessary in order to facilitate both qualitative and quantitative analyses.

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3. Materials and Methods

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3.1. Bacterial strains and plasmids

The bacterial strains and plasmids used in this work are reported in Tables 3.1 and 3.2 respectively. E.coli JM109 [88] was routinely used for plasmid construction and selection.

Plasmids were isolated using standard procedures [89] or by use of purification kits (QUIAGEN) and introduced into the bacterial host by electroporation [90].

Strain Features Reference

Pseudomonas sp. N1 Able to use naphthalene as the sole carbon and energy source

This study Pseudomonas sp. N2 Able to use naphthalene as the sole carbon

and energy source

This study Sphingobium sp. PhS Able to use phenanthrene as the sole carbon

and energy source

[91]

E.coli JM109 recA1, endA1, gyrA96, thi, hsdR17[rk+

mk+], supE44, relA1 λ-∆ [lac-proAB], F’

[traD36, proAB+, lacIqZ∆M15]

[88]

E. coli ER2566 F- λ- fhuA2 [lon] ompT lacZ::T7 gene 1 gal sulA11 Δ(mcrC-mrr)114::IS10 R(mcr- 73::miniTn10-TetS)2 R(zgb-

210::Tn10)(TetS) endA1 [dcm]

IMPACT™-CN, N.E.

BioLabs, Inc

3.2. Culture methodology

3.2.1. Bacterial strains isolated from enrichment cultures

Bacterial strains were grown in Luria Broth (LB) [89] or M9 mineral medium (M9) [92] at 30 °C. In addition, M9 minimum medium, an M9/glucose solution 0.2% (w/v), was also used.

Biphasic system: the biphasic system consisted of M9 mineral medium as the water phase, and Dibutyl Phthalate (DBP) as the organic phase. The appropriate carbon source was added to DBP at a concentration of 400 mg/l; cultures were prepared by adding cells previously grown in M9 minimum medium to a mixture composed of 10% substrate/DBP solution and 90% M9, and incubated at 30 °C. The increase in biomass was evaluated using spectrophotometer readings at a wavelength of 600 nm.

Table 3.1. Bacterial strains.

Bacterial oxygenases: potential, features and new investigation methods.

UNIVERSITY OF INSUBRIA

Ph.D. School in Biological and Medical Sciences

- XXIV Course -

BACTERIAL OXYGENASES:

FEATURES, POTENTIAL

AND NEW INVESTIGATION METHODS

2008-2011

Table of Contents

Abstract

1. Introduction

b a

2. Aims

3. Materials and Methods